Target-specific proteins have been shown to specifically localize and bind to pre-selected target sites, when introduced into an organism. These proteins can be used as carriers of diagnostic and therapeutic agents for specifically localizing such agents to the target. The use of target-specific proteins as carriers involves the binding of the diagnostic or therapeutic agent to the protein to form a conjugate which has the diagnostic or therapeutic properties of the diagnostic or therapeutic agent, and the localization properties of the protein.
The use of monoclonal antibodies and antibody fragments as target-specific carriers of diagnostic or therapeutic agents provides an efficient means of localizing such agents to target tissue. Monoclonal antibodies are highly specific and can be used, for example, for imaging specific target sites or as vehicles to deliver substances to target sites. In recent years numerous monoclonal antibodies have been developed with affinity for targets such as atherosclerotic tissue, fibrin, myosin and tumors, for example. The attachment of radiometals to proteins, especially antibodies and antibody fragments, results in the formation of new radiodiagnostic and radiotherapeutic agents.
Proteins and antibodies have been shown to form stable bonds to radiometals by the use of bifunctional coupling agents. A suitable bifunctional agent is capable of binding radiometals by chelation and can also form a stable linkage to the protein. Thus, the protein or antibody is bound to the radiometal through the bifunctional coupling agent. For example, diethylenetriaminepentaacetic acid (DTPA) has been conjugated onto an antimyosin antibody, and the protein-bound DTPA used to chelate indium-111. See Khaw, et al., Science, 209, 295-97 (1980); Krejcarek, et al., Biochem. Biophys. Res. Comm., 77, 581-85 (1977) and Childs, R. L. and Hnatowich, D. J., J. Nucl. Med., 26, 293 (1985). This approach has also been used where particular diaminodithiol and diamidedithiol chelating agents have been coupled to antibodies and antibody fragments. See Fritzberg, et al., J. Nucl. Med., 27, 957-58 (1986), Eary, J. et al., J. Nucl. Med., 28, 650-51 (1987) and A. R. Fritzberg, et al., Proc. Nat. Acad. Sci. USA, 85, 4025-29 (1988)).
Chelated radiometals and bifunctional coupling agents have been linked to proteins by lysyl side chain amino groups. See EPO Publication No. 188, 256. Chelators have also been site selectively attached to oxidized antibody carbohydrate moieties. See EPO Publication No. 173, 629 and U.S. Pat. No. 4,671,958. Chelators can also be attached by reaction with free sulfhydryl groups. See, for example, U.S. Pat. No. 4,659,839, U.S. Pat. No. 4,671,958 and EPO Publication No. 173,629.
Antibodies and antibody F(ab').sub.2 fragments are symmetrical proteins containing two identical binding sites. The binding of antibodies and antibody F(ab').sub.2 fragments to a target is enhanced relative to the Fab', Fab and Fv fragments which contain only a single binding site. It is believed that the F(ab').sub.2 fragment may be superior to the whole antibody for the preparation of radioimmunotherapeutic and especially radioimmunodiagnostic agents due to a shorter half-life in vivo. See, for example, J. Shani, et al., Nucl. Med. Biol., 16, 33-40 (1989). Further, the absence of the Fc portion of the antibody which can interact non-specifically with Fc receptors may provide additional benefits. See, for example, R. L. Wahl, et al., J. Nucl. Med., 24, 316-25 (1983). The radiolabeled F(ab').sub.2 fragment has been shown to be superior to either IgG or Fab for delivery of activity to tumor sites in models of radioimmunotherapeutic applications. See, for example, R. Sutherland, et al., Canc. Res., 47, 1627-33 (1987), K. Z. Walker, et al., Nucl. Med. Comm., 9, 517-26 (1988).
The F(ab').sub.2 fragment is composed of two identical heavy chain fragments linked by one or more disulfide bridges and two identical light chains each linked to one heavy chain by a disulfide bridge. Exposure of the F(ab').sub.2 fragment to suitable reducing conditions cleaves the interchain disulfide bonds to free sulfhydryls. This reduction gives two identical Fab' fragments, each consisting of a heavy chain fragment and a light chain held together by noncovalent interactions. Each Fab' fragment also contains a single binding site. Examples of reducing conditions that can effect this cleavage include thiols (sulfhydryls) and other reducing agents such as tin (II).
The use of F(ab').sub.2 fragments to prepare radioimmunotherapeutic and radioimmunodiagnostic agents is believed to be superior to whole IgG or monovalent fragments in some instances. The disulfide bond joining the two halves of the F(ab').sub.2 fragment is, however, susceptible to cleavage. There are instances of F(ab').sub.2 cleavage to Fab' in vivo, thus eliminating the advantages of the F(ab').sub.2. See, for example, S. E. Halpern, et al., J. Nucl. Med., 28, 692 (1987). The use of certain radiometals for the formation of radioimmunotherapeutic or radioimmunodiagnostic agents require the use of reducing conditions to adjust the oxidation state of the radiometal. These conditions, when applied to a F(ab').sub.2, can lead to preferential formation of radiolabeled Fab', again precluding the advantages of the F(ab').sub.2. See, for example, A. M. Zimmer, et al., Cancer Res., 47, 1691-94 (1987). In addition, a radiometal chelator linked to a protein by a disulfide will undergo cleavage in vivo. See C. F. Meares, et al., Int. J. Cancer, Supp 2, 99 (1988). The potential advantage of the two binding sites of the F(ab').sub.2 fragment is lost in the situations where reductive cleavage of the interchain disulfide bond occurs.
Reagents have also been described for the covalent linkage of two proteins. Homobifunctional linking reagents, that is linking reagents containing two identical reactive sites, have also been described for cross-linking proteins. The most common examples contain two amine-selective reactive sites or two sulfhydryl selective reactive sites. For example, N,N'-1,4-phenylenedimaleimide has been used to cross-link enzymes and antibodies in the preparation of reagents for enzyme-linked immunoassays. Bis-N-maleimidomethyl ether and N,N'-1,2-phenylenebismaleimide have both been used to prepare bispecific thioether-linked F(ab')2 fragments. See M. J. Glennie, et al., J. Immunol., 139, 2367-75 (1987) and J. M. Frincke, et al., J. Nucl. Med., 692 (1987). These bispecific thioether-linked F(ab')2 fragments consist of two non-identical Fab1 fragments with different binding specificities cross-linked via their sulfhydryls by the bis-maleimide homobifunctional reagent.
In spite of these disclosures, there remains a need for a cross-linked protein composition having at least two identical binding sites, which is suitable for use in immunodiagnostic and immunotherapeutic applications. There is also a need for a simple method of making this protein composition.